The innate and adaptive arms of the immune system are integrally linked, with dendritic cells (DCs), innate antigen presenting cells, providing critical instructions to elicit the generation of adaptive T cell immunity. Using a novel analytical imaging approach, we recently uncovered that different DC populations are asymmetrically distributed in steady-state mouse lymph nodes, creating highly segregated zones composed of one or another DC subset. Given that these DC populations have well-characterized differences in their abilities to elicit distinct types of T cell responses, these findings raise the central hypothesis of this proposal, that lymphoid tissues are composed of a mosaic of previously unappreciated DC microenvironments that are uniquely dedicated for the generation of specific programs of T cell immunity. The objective of this grant is to characterize these innate cell microenvironments during the steady-state and inflammation in both mice and humans, and to study how they influence adaptive immune responses to vaccines. We will accomplish this goal in three Specific Aims. In Aim 1, we will perform a comprehensive characterization of DC microenvironments in mouse and human lymph nodes during the steady-state and after induction of inflammation with distinct vaccine adjuvants. In Aim 2, we will investigate the molecular mechanisms regulating DC positioning in lymphoid tissues. In Aim 3, we will interrogate how the spatial organization of DCs influences CD4+ and CD8+ T cell responses to vaccines. Our rationale is that a better understanding of lymphoid tissue microanatomy, with respect to innate and adaptive immune cell crosstalk, will help reveal the underlying principles of immune response generation to vaccines. A long-term goal of our studies is to develop novel approaches to manipulate in vivo cellular positioning in order to better modulate and fine-tune the innate and adaptive response to vaccines. In summary, the proposed studies are significant, as they will advance our basic understanding of how immune responses are generated in vivo, while also promoting development of novel strategies for immunomodulatory therapeutics and vaccines.